Alternative microbial-based functional ingredient source for lycopene, beta-carotene, and polyunsaturated fatty acids

The acquisition of carotenoids and polyunsaturated fatty acids (PUFAs) from plants and animals for use as functional ingredients raises concerns regarding productivity and cost; utilization of microorganisms as alternative sources is an option. We proposed to evaluate the production of carotenoids and PUFAs by Rhodopseudomonas faecalis PA2 using different vegetable oils (rice bran oil, palm oil, coconut oil, and soybean oil) as carbon source, different concentrations of yeast extract as nitrogen source at different cultivation time to ensure the best production. Cultivation with soybean oil as source of carbon led to the most significant changes in the fatty acid profile. Compared to the initial condition, the strain cultivated in the optimal conditions (4% soybean oil, 0.35% yeast extract, and 14 days of incubation) showed an increase in μmax, biomass, carotenoid productivity, and microbial lipids by 102.5%, 52.7%, 33.82%, and 34.78%, respectively. The unsaturated fatty acids content was raised with additional types of PUFAs; omega-3 [alpha-linolenic acid and eicosapentaenoic acid] and omega-6 [linoleic acid and eicosatrienoic acid] fatty acids were identified. The results of ultra high-performance liquid chromatography-electrospray ionization-quadrupole time of flight-mass spectrometry (UHPLC-ESI-QTOF-MS/MS) indicated the molecular formula and mass of bacterial metabolites were identical to those of lycopene and beta-carotene. The untargeted metabolomics revealed functional lipids and several physiologically bioactive compounds. The outcome provides scientific reference regarding carotenoids, PUFAs, and useful metabolites that have not yet been reported in the species Rhodopseudomonas faecalis for further use as a microbial-based functional ingredient.


Introduction
Increasing concern for health and well-being has increased the inclusion of functional ingredients in dietary supplements, nutraceuticals, and health products. Carotenoids and polyunsaturated fatty acids (PUFAs) are examples of high-value compounds supplemented in many products. Carotenoids, natural biomolecules produced by plants, algae, and some bacteria, have been shown to have provitamin A activities and strong antioxidant potential, allowing them to fight cancer, age-related macular degeneration, photooxidative damage, and boost immunological response [1]. Carotenoids have received interest in the nutraceuticals and food industries, with a market worth of around 1.21 billion USD [2,3]. Functional lipids are compounds involved in a broad spectrum of metabolic conditions. Long-chain PUFAs in n-3 and n-6 series (omega-3 and omega-6 fatty acids) have been discovered as the essential fatty acids in mammals because of their specific biofunctions as precursors for eicosanoids that modulate pulmonary function [4], structural components of membranes, and inflammatory responses [5]. There is plenty of data to show that PUFAs can help avoid a variety of chronic diseases [6]. Alpha-linolenic acid (18:3, n-3; ALA), eicosapentaenoic acid (20:5, n-3; EPA), docosahexaenoic acid (22:6, n-3; DHA), linoleic acid (18:2, n-6; LA), and arachidonic acid (20:4, n-6) are all examples of essential fatty acids. Carotenoids and PUFAs cannot be biosynthesized by human body, making them the crucial functional ingredients in several products.
There are many carotenoid-based products on the market, as well as dietary supplements with PUFAs [7,8]. Carotenoids and PUFAs derived from plants and animals raise concerns not only their productivity but also production cost. PUFAs are found in high-price food such as chia seeds, fish oils, and marine fish in the families Scombridae, Clupeidae, and Salmonidae [9]. Plant carotenoids require agricultural land, pesticides, growing season, and harvesting time. Therefore, carotenoids and PUFAs acquire from these sources as the functional ingredients are expensive. At present, the use of microbial biomass as a functional ingredient is a viable option to provide key nutrients at a cheaper cost with a higher yield [10]. As a result, microorganisms are increasingly used in functional food, functional ingredients, and nutraceuticals businesses. Carotenoids and PUFAs have been acquired from a variety of bacteria, fungi, and microalgae for use as active ingredients in industries. Beta-carotene from Sphingomonas sp. and canthaxanthin from Paracoccus carotinifaciens are examples of bacterial carotenoids used in food colorants [3] while lycopene from Rhodospirillum rubrum and spheroidenone from Rhodobacter sphaeroides have been shown to have anti-cancer and anti-inflammatory properties in health products [11]. Nutritional supplements containing beta-carotene and astaxanthin derived from the algae Dunaliella salina and Haematococcus pluvialis are also reported [12]. Mortierella alpina, Mortierella alliacea [6], and Rhodotorula mucilaginosa are among the fungal producers of omega-3 and omega-6 fatty acids [13]. A heterotrophic unicellular marine thraustochytrid Aurantiochytrium sp. [14]. and a microalga Crypthecodinium cohnii [15] have been used as sources of DHA and squalene. Several significant metabolites from microorganisms are currently investigated to explore their application and utilization as functional ingredients.
The anoxygenic photosynthetic bacteria are excellent producers of carotenoids and PUFAs. Because of their several modes of metabolism, these bacteria have been widely used in waste treatment. They are not pathogens but they do contain several types of useful compounds such as coenzyme Q 10 , 5-aminolevulinic acid, carotenoids, bacteriochlorin, and polyhydroxyalkanoates [16]. They have membrane lipids and phospholipids which are not typically found in general bacteria, such as phosphatidylcholine, sulfoquinovosyldiacylglycerol, betaine lipids, and ornithine lipids [17]. Although their prominent characteristics have been reported to be used as single-cell protein (SCP) and feed [18], they have not yet received the attention they deserve, and the information about utilization of these bacteria as functional ingredients is scarce. Lycogen™, a carotenoid product acquired from Rhodobacter sphaeroides WL-APD911, is the only product obtained from anoxygenic photosynthetic bacteria utilized in mammals that shows anti-inflammatory, anti-oxidative, and glucose homeostasis effects [11,19].
The anoxygenic photosynthetic bacterium Rhodopseudomonas faecalis PA2 contains several nutrients [20] and high protein content containing all essential amino acids [21] although it was cultivated on waste substrates. Aquatic animals fed R. faecalis PA2 showed superior performances and survival in comparison with animals fed the alga Chlorella vulgaris, the yeast Saccharomyces cerevisiae, the cyanobacterium Spirulina sp., and the other species of anoxygenic photosynthetic bacteria [22,23]. This indicates it could be a potential candidate for application in functional ingredient industries and the investigation of additional useful metabolites of this strain has drawn attention.
To produce the microbial-based functional ingredients, carbon source for microbial growth is crucial and the expense of carbon source has to be factored in. Organic acids, such as malic acid and succinic acid, are the essential carbon for anoxygenic photosynthetic bacteria but they are the expensive feedstock. On the other hand, vegetable oils are much cheaper; the catabolism of oil components produces organic acids as intermediates that can be used for the growth of anoxygenic photosynthetic bacteria [24]. Hence, the objectives of this study were to identify lycopene, beta-carotene, and PUFAs in the anoxygenic photosynthetic bacterium R. faecalis PA2 in the presence of vegetable oils and to evaluate the metabolite profiling of this strain. In this study, a Liquid Chromatography-Mass Spectrometry (LC-MS)-based metabolomic approach was used to investigate the metabolic composition, aiming to reveal the interesting metabolites in this strain. To the best of our knowledge, this is the first study that used metabolomics to quantify the useful metabolites and to observe the metabolites which have not been reported in the anoxygenic photosynthetic bacteria.

Effects of vegetable oils as carbon sources on biomass, carotenoids, microbial lipids, and fatty acid composition
The photosynthetic bacterium R. faecalis PA2 was employed which is safely deposited at Thailand Bioresource Research Center (TBRC 5694) for research and commercial purposes. The cultivation of this strain and inoculum preparation were carried out in glutamate-malate medium and exposed to light intensity at 4000 lux under anoxygenic conditions [25]. The basal medium (BM) supplemented with 1% vegetable oil as a carbon source (rice bran oil, palm oil, coconut oil, or soybean oil) was used as the tested medium and adjusted pH to 6.8; inoculum volume was 10%. Incubation was carried out at 30 ± 2 • C under light-anoxygenic conditions. The experiments were conducted in six replicates. Biomass, carotenoid, and microbial lipid concentrations were investigated at intervals of 48 h. Bacterial cells were separated from the culture broth by centrifugation at 6000 rpm 4 • C for 10 min at the end of the experiment (Himac CR20B2, Hitachi, Tokyo, Japan). The supernatant was discarded; the cell pellets were washed with 0.85% sterile NaCl and then freeze-dried using a freeze dryer (Freezone 2.5 L; LABCONCO, KC, USA). The fatty acid composition of the freeze-dried biomass was determined following AOAC [26] method 996.06. Briefly, the Shimadzu Nexis GC-2030 equipped with split injector port, flame ionization detector (FID), and AOC-20i + s autosampler was used. The fatty acid methyl ester (FAME) mix was analyzed according to the AOAC method 996.06 which required the use of helium carrier gas (constant linear velocity 18 cm/s). The column was Rt-2560 100 m × 0.25 mm ID × 0.20 μm film thickness. The GC parameters included inlet (1 μL split injection; 225 • C; split ratio 200:1) and flame ionization detector (285 • C; H 2 32 mL/min; air 200 mL/min; make-up (N 2 ) 24 mL/min). The oven temperature was 100 • C (4 min hold); 3 • C/min to 240 • C (15 min hold). The FAME mix was purchased from Restek (PA, USA).

Optimization of cultural condition
Since carbon content, nitrogen (yeast extract) content, and incubation period play the significant roles in boosting bacterial growth and essential metabolites, these three parameters were investigated. The optimization was carried out by one-variable at a time. The optimal vegetable oil was used as a carbon source in BM. The vegetable oil content (1%, 2%, 4%, 6%, 8%, and 10% (w/v)) was supplemented in BM and adjusted pH to 6.8. The 10% inoculum was included. The experiment was set for 10 days at 30 ± 2 • C under light-anoxygenic conditions. The biomass, carotenoid, and microbial lipid concentrations were investigated at intervals of 48 h. Six duplicates of each experiment were carried out. For the optimization of yeast extract content, the contents of 0.05%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.80%, and 1.60% were optimized. The incubation period of 6, 8, 10, 12, and 14 days were investigated. The incubation conditions were carried out as stated.

Bacterial cultivation under the optimal conditions and determination of fat and fatty acid composition
Rhodopseudomonas faecalis PA2 was grown in a photo-bioreactor with 10% inoculum under optimal conditions. Nitrogen gas was flushed into the reactor to create an anoxygenic condition. Illumination (4000 lux) was provided throughout the experiment. Bacterial cells were freeze-dried and used to determine total fat and fatty acid composition by the hydrolytic extraction gas chromatographic technique.

Cell extraction for metabolite measurement
The wet cells were used, and 50 mg of the sample (five replicates) was dissolved with 1 mL reconstitution buffer (water: acetonitrile = 1:1). The mixture was sonicated for 15 min three times (Ultrasonic Cleaner GT SONIC-D2, GT SONIC, Shenzhen, China) and centrifuged at 15 000 rpm at 4 • C for 15 min twice (D3024R High Speed Refrigerated Micro Centrifuge, DLAB, DLAB Scientific, Beijing, China). The supernatant was transferred to the high-performance liquid chromatography (HPLC) glass vial for LC-MS data acquisition.
The mass spectrometry was performed using the broadband collision-induced dissociation (bbCID) method by a compact ESI-Q-TOF system (Bruker Daltonics, Bremen, Germany). Sodium formate solution (2 mM sodium hydroxide, 0.1% FA, 50% isopropanol) was injected as an external calibrant with a flow rate of 0.5 μL/min. The condition in positive ionization polarity mode consisted of 50-1300 m/z mass range, 35 V cone voltage, 4000 V capillary voltage, 220 • C source temperature, 220 • C desolvation temperature, and 8 L/min desolvation gas flow.
For the untargeted metabolite profiling analysis, the flow rate was adjusted to 0.35 mL/min; the gradient elution was set as follows: ionization polarity mode are 50-1300 m/z mass range, 35 V cone voltage, 4000 V capillary voltage, 220 • C source temperature, 220 • C desolvation temperature, and 8 L/min desolvation gas flow. The conditions in negative ionization polarity mode are 50-1300 m/z mass range, 31 V cone voltage, 4500 V capillary voltage, 220 • C source temperature, 220 • C desolvation temperature, and 8 L/min desolvation gas flow.

Metabolite identification and annotation
The data was imported to the MetaboScape software for metabolite identification. The assessment of metabolites was compared with the public database: METLIN, Human Metabolome Database (HMDB), Bruker MetaboBASE, and LipidBlast database. Level of assignment (LoA) of the metabolites include 1) accurate mass matched to the database, 2) accurate mass matched to database and tandem MS spectrum matched to in silico fragmentation pattern, 3) tandem MS spectrum matched to database or literature, 4) retention time and the molecular mass matched to standard compound, and 5) MS/MS spectrum matched standard compound.

Analytical procedures
Bacterial biomass and carotenoids were analyzed according to Saejung and Chanthakhot [25]. Carotenoids were extracted by immersing the cell pellets in methanol-acetone (2:3 v/v) solution overnight until the colorless cells were obtained. The pigment extract was read at 480 and 770 nm using a Genesys 20 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Microbial lipids were  extracted from cells by centrifugation of the culture broth at 9000 rpm 4 • C for 15 min. The pellets were resuspended in distilled water after being washed twice with 0.9% NaCl. The pellets were boiled for 10 min in 1 N NaOH, and the cell debris was discarded [27]. The supernatant was used to examine microbial lipids by saponification with 1.5 M KOH in 80% ethanol following Kwon and Rhee [28].

Statistical analyses
The data were presented as mean ± standard deviation (SD). The significant differences between means were calculated by oneway analysis of variance (ANOVA). The Duncan's multiple range test was used to compare the means at a significance level of p ≤ 0.05. The software IBM SPSS Statistics 28.0.0.0 (IBM Corp., Armonk, NY, USA) was used for statistical analyses.

Effects of vegetable oils as carbon sources on biomass, carotenoids, microbial lipids, and fatty acid composition
All the tested vegetable oils could be used as sole carbon-based nutrients. This phenomenon is supported by Fig. 1a-d, which depicts the growth of R. faecalis PA2 and the generation of some metabolites in the presence of vegetable oils. The use of coconut oil as a carbon source resulted in the lowest maximum specific growth rate (μ max ) (0.082 ± 0.005/day), carotenoid concentration (452.58 ± 8.56 mg/ L), and microbial lipid concentration (134.28 ± 7.66 mg/L). Soybean oil, on the other hand, showed the highest values. The biomass of R. faecalis PA2 fed coconut oil had the highest saturated fatty acid content (Table 1). However, cultivation with soybean oil showed the predominant fatty acid composition because the biomass of R. faecalis PA2 contained both omega-3 fatty acid (ALA) and omega-6 fatty acids (LA and eicosatrienoic acid [or dihomo-gamma-linolenic acid (DGLA]). Therefore, soybean oil was employed in the following experiments due to the composition of unsaturated fatty acids.

Optimization of soybean oil contents
As shown in Fig. 2a, as the content of soybean oil increased, μ max increased, with the maximum level of 0.64 ± 0.01/day, occurring at 4%, above which the μ max decreased. Fig. 2b-d shows that supplementing with 4% soybean oil resulted in the highest biomass, carotenoid, and microbial lipid concentrations; however, there was no statistical difference of microbial lipids among the treatments. Table 2 indicates the μ max at 4% soybean oil was enhanced by 60% when compared to the initial condition (1% soybean oil).

Optimization of yeast extract content
Yeast extract is the most effective nitrogen source for R. faecalis PA2 [29], thus, the optimal content should be investigated. The μ max and biomass concentration varied depending on yeast extract content ( Fig. 3a and b). The highest μ max and biomass concentration were found at 0.35% yeast extract, with the increase by 135% and 73.65%, respectively (Table 2). Carotenoid synthesis is reduced at lower C/N ratios (0.80%-1.60% yeast extract) (Fig. 3c). The concentration of microbial lipids was dramatically reduced when yeast Table 2 Comparison of μ max , biomass concentration, carotenoid productivity, and microbial lipid concentration of Rhodopseudomonas faecalis PA2 grown in each optimization study and initial condition.  extract content was greater than 0.35% because of the excessive nitrogen level (Fig. 3d).

Optimization of the incubation period
The μ max , biomass concentration, carotenoids, and microbial lipid production increased with increasing incubation period, as indicated in Fig. 4a-d, with the maximum at 14 days. In comparison to the initial condition, carotenoids were increase by 33.82% (Table 2). A long incubation period boosted carotenoid production because they are produced during stationary phase. An increase in carotenoids was found after 12 days of incubation (Fig. 4C).

Determination of fatty acid composition
The kinetic characteristics of R. faecalis PA2 acquired from a photo-bioreactor are presented in Table S1. Table 3 indicates the total fat and fatty acid composition of R. faecalis PA2 cultivated in the optimal conditions compared with the initial condition. The proposed conditions (4% soybean oil, 0.35% yeast extract, and 14 days of incubation) could enhance the content of unsaturated fatty acid significantly compared with the initial condition. The unsaturated fatty acids cis-10-pentadecenoic acid (15:1, n-10), cis-10-heptadecenoic acid (17:1, n-10), and EPA were present in the optimal conditions, whereas they were not found in the initial condition (Table 3). This study revealed that ALA, EPA, LA, and DGLA were found in the biomass of R. faecalis PA2 grown in the proposed conditions. To our knowledge, EPA was first reported in this species. Therefore, using soybean oil as carbon source along with the conditions described in this study might provide the important PUFAs in R. faecalis PA2.

Determination of carotenoids and the untargeted profiling of metabolites using UHPLC-ESI-QTOF-MS/MS
Lycopene and beta-carotene are dietary carotenoids found in fruits and vegetables. They play a vital role in providing health benefits due to their anti-oxidant properties [30]. Therefore, these two carotenoids were quantified using UHPLC-ESI-QTOF-MS/MS-based targeted metabolomics. Fig. 5a and b shows the extracted ion chromatograms (EIC) of the standard carotenoids compared with the samples. The molecular formula and mass of the samples were identical to that of the standard lycopene and beta-carotene. The adduct ions were [M+H] + which indicated that the additional molecule was the proton. The   10) 0.020 ± 0 a ab Palmitoleic acid (16:1, n-7) 0.407 ± 0.05 a 0.726 ± 0.71 a cis-10-Heptadecenoic acid (17:1, n- 10) 0.053 ± 0.01 a ab cis-9-Oleic acid (18:1, n-9) 7.491 ± 1.11 a 2.123 ± 0.07 b cis-9,12-Linoleic acid (18:2, n-6) c 0.084 ± 0.04 a 1.440 ± 0.23 a alpha-Linolenic acid (18:3, n-3 Table 4. Although the mass of lycopene and beta-carotene was identical, the time that they were eluted was different which was used to identify the carotenoid type. The molecular formula and exact mass of the detected samples showed that this strain contained lycopene and beta-carotene. As far as we know, beta-carotene has not been reported in the species Rhodopseudomonas faecalis. To the best of our knowledge, this is the first study to report beta-carotene and lycopene in R. faecalis verified by the targeted metabolomic analysis. The untargeted metabolite profile chromatogram of the samples (five replicates) in positive ionization mode is shown in Fig. 6. The five samples showed identical peaks and there were several metabolites found in this train in the untargeted mode. Each metabolite was identified by comparing with the public database. The identified metabolites are presented in Table 5; the relative concentration of all metabolites was quantified with the external calibrants. Table 6 summarizes the useful metabolites acting as functional ingredients or physiologically bioactive compounds and their benefits. The results also showed several types of phospholipids found in egg yolk, meat, and nuts including lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE), and phosphatidylcholine (PC or lecithin) ( Table 6). A microbially associated metabolite, desaminotyrosine, has been found. Buddledin A, (E)-2-octenyl butyrate, and piperonyl acetate were also detected in the cells of R. faecalis PA2 cultivated in soybean oil under the optimal conditions.

Discussion
Many studies have been conducted to explore the alternative organisms for the production of carotenoids and PUFAs replacing the production from plants and animals. The use of microorganisms to produce these compounds has increased significantly; yet, few investigations have been undertaken to uncover additional carotenoids and PUFAs producers. In this study, a strategy that used soybean oil as feedstock to produce functional ingredients from beneficial bacterium was established. The strain was able to use vegetable oils by digesting them into glycerol and fatty acids. The glycerol is transformed into dihydroxyacetone phosphate, one of the glycolysis intermediates, and receives energy in the form of ATP through the metabolic process [24]. The fatty acids are metabolized via beta-oxidation, which produces either acetyl Co-A or succinyl Co-A depending on the type of fatty acids. Acetyl Co-A is required in the TCA cycle's transition reaction to combine with oxaloacetic acid while succinyl Co-A is one of the intermediates in the TCA cycle, thus, the strain can generate energy by using fatty acids as a carbon source [31]. Rice bran oil, palm oil, coconut oil, and soybean oil have 25%, 49.3%, 83%, and 15% saturated fatty acids, respectively [32,33]. Coconut oil contains the highest content of saturated fatty acids. The saturated fatty acids have higher melting points than unsaturated fatty acids, resulting in requiring more metabolic energy to break down [34]. Photosynthetic bacteria prefer organic acids for growth while coconut oil contains only trace amounts of free fatty acids, thereby influencing the degradation by this strain. Moreover, coconut oil was found to be resistant to microbial degradation in other study [35]. Soybean oil contains 81% unsaturated fatty acids [32], which resulted in facilitating the catabolism by bacteria.
Since multiple intermediates are involved in carotenoid biosynthesis, the important precursor to manufacture these intermediates is acetyl-CoA [36]. As a result, acetyl Co-A acquired from the breakdown of vegetable oils can be employed as a precursor for carotenoid synthesis (Fig. 1c). Moreover, acetyl-CoA is used as a precursor to producing malonyl Co-A via carboxylation and then transformed to acetyl-ACP by transacylase for use in lipid biosynthesis. The produced lipids are then transported and stored in bacterial cells [37], hence using vegetable oils as carbon sources aided the buildup of microbial lipids in bacteria. This was in line with prior research reporting the supplementation of phototrophic microorganisms with carbon precursors could increase lipid accumulation [38].
In this study, soybean oil was the only vegetable oil that could boost ALA in the tested strain (Table 1). This was because soybean oil is categorized as an alpha-linolenic acid oil, which contains a significant amount of ALA [39]. ALA, EPA, and DHA are the three important omega-3 fatty acids; DHA and EPA are found in fish and seafood. ALA, on the other hand, can be transformed into EPA and ultimately to DHA [40].
As shown in Fig. 2, μ max , carotenoids, and microbial lipid were increased at a certain concentration of soybean oil. This was likely because the high content of carbon source increased the carbon skeleton for the biosynthetic pathway, leading to enhance microbial growth and metabolites [41]. Moreover, the carbon to nitrogen (C/N) ratio of the medium is involved because the greater C/N ratios increase lipid and carotenoid synthesis [42]. Excess carbon supply, on the other hand, caused a decrease in growth rate due to substrate inhibition [43].
The C/N ratio was inversely proportional to the amount of yeast extract present (Fig. 3). When compared to the experiment supplemented with 0.35% yeast extract, the concentrations of yeast extract ranging from 0.05% to 0.30% had a greater C/N ratio. The differences in microbial biomass and respiration reflected these differences. The experiment fed a little amount of yeast extract resulting in the deficiency of nitrogen for biosynthetic pathways at the same amount of carbon. Despite the availability of carbon, the anabolic process comes to a halt. Bacterial growth with a higher C/N ratio is confronted with a surplus of C to N, whereas growth with a lower C/N ratio is confronted with a lack of C to N [44]. As shown in Fig. 4, the longer incubation period resulted in higher biomass, which led to more lipids and carotenoids in bacterial cells. Under the optimal conditions (4% soybean oil, 0.35% yeast extract, and 14  days of incubation), the lipid productivity was 13.86 mg/L/day (Table S1), whereas lipid productivity of Chlamydomonas reinhardtii, Chlorella sorokiniana, and Scenedesmus obtusus XJ-15 were 10.9 mg/L/day, 0.502 mg/L/day, and 0.607 mg/L/day, respectively [45][46][47]. Previous research reported that carotenoid productivity of Dunaliella tertiolecta, Chlorella vulgaris UTEX 265, and Scenedesmus sp. were 0.86 mg/L/day, 11.98 mg/L/day, and 19.70 mg/L/day, respectively [48][49][50] while carotenoid productivity of R. faecalis PA2 was 45.37 mg/L/day (Table S1). It can be concluded that the lipid productivity and carotenoid productivity of this strain were comparable with the other photosynthetic microorganisms.
The results of this study also prove that changes in the medium composition produce quantitative alteration in fatty acids and carotenoids of anoxygenic photosynthetic bacteria. A previous study showed that the fatty acid composition of bacteria is regulated by the medium composition as well as the age of the cells [51]. This study also calculated the relationships between the ratio of unsaturated to saturated fatty acids (UFA:SFA ratio) because it can be used to evaluate fat utilization. Fat utilization increased with the increase in UFA:SFA ratio; reaching a maximum at UFA:SFA ratio of 4 [52]. Obviously, the strain grown in the optimal condition provided a greater UFA:SFA ratio compared with the initial condition (Table 3). Moreover, previous work also reported the advantage of high UFA:SFA ratio in animal diets in improving meat quality [53]. Omega-6 (LA) can be converted to omega-3 (ALA); thus, the enzymes involved in the metabolism of omega-3 and omega-6 fatty acids are shared and they regulate each other. The balance of omega-6/omega-3 fatty acids in the diet is vital for human nutritional needs. Excessive amount of omega-6 or high omega-6 to omega-3 ratio can cause pathogenesis of diseases [54]. The proportions of omega-6 and omega-3 in the diet can predict the biochemical efficiency, approaching the ratio of 2:1 or 1:1 omega-6/omega-3 fatty acids are the ideal for health. As shown in Table 3, the omega-6/omega-3 fatty acids ratio of R. faecalis PA2 cultured in the optimal condition was close to the targeted ratio. R. faecalis PA2 contained several metabolites found in foods originating from plants and animals. The detection of functional lipids ALA, EPA, LA, and DGLA in biomass has drawn attention because of their physiological and structural roles in biological systems as shown in Table 6. These metabolites are recognized as high-value compounds supplemented in dietary supplements [56,62], suggesting that the biomass of R. faecalis PA2 can be utilized as an alternative source for MUFAs and PUFAs.
The results of UHPLC-ESI-QTOF-MS/MS analysis ensured that the strain and the proposed conditions produced beta-carotene and lycopene. The additional metabolites were detected in cells. The untargeted metabolomics analysis revealed the other functional lipids such as phosphatidylcholine which is a multifunctional phospholipid required for the incorporation of cholesterol in membranes [63]. Our results also showed the presence of desaminotyrosine in R. faecalis PA2. According to previous study, this metabolite can protect against influenza virus [65] and maintain systematic immune homeostasis [66]. Nα-acetyl-L-glutamine can be supplemented in sports nutrition's products to help boost exercise endurance and prevent the negative effect of protein energy malnutrition [68]. Buddledin A showed an antifungal effect [67]. (E)-2-octenyl butyrate is used as a flavoring ingredient, whereas piperonyl acetate is found in the green vegetables [69,70]. In our perspective, R. faecalis PA2 cultured under the aforementioned conditions could be an alternative source for microbial-based functional ingredients. Although R. faecalis PA2 could provide several beneficial metabolites and it is a promising source for alternative microbial-based functional ingredient, further investigation in vivo is required to verify its safety and efficiency before practical application.

Author contribution statement
Chewapat Saejung: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials and analysis tools; Wrote the paper.Khomsorn Lomthaisong: Contributed reagents and analysis tools. Prawphan Kotthale: Performed the experiments; Analyzed and interpreted the data.

Table 6
Summary of the useful metabolites of Rhodopseudomonas faecalis PA2 cultivated in the optimal conditions.

Metabolite
Applications Reference Lycopene Nutrient supplement used as antioxidant, anti-cancer, and anti-inflammatory properties.
[11] Beta-carotene Nutrient supplement for adult and infant foods which is used as vitamin A precursor.
[ [60][61][62] Phosphatidylcholine and Phosphatidylethanolamine (PE) Improvement of EPA and DHA levels in brain that can enhance the treatment of depression and neuroinflammatory diseases such as Alzheimer's disease. Reducing atherosclerosis by decreasing plasma very low-density lipoprotein-cholesterol (VLDL-C) and increasing plasma high-density lipoprotein-cholesterol (HDL-C) [63,64] Desaminotyrosine Protection of influenza virus infection through modification of type I interferon signaling and diminution of lung immunology and acting as an anti-inflammatory molecule that contribute to maintain intestinal and systematic immune homeostasis. [65,66] Buddledin A Antifungal action against Trichophyton rubrum, Tricophyton interdigitale, and Epidermophyton floccosum [67] Nα-Acetyl-L-glutamine Prevention of gut damage induced by protein energy malnutrition.